Ikaros mutant mice show multiple hematopoietic defects, particularly in the lymphoid lineages. In humans, aberrations in Ikaros expression have been linked to leukemia. The proposed project will examine the expression of Ikaros isoforms in normal human hematopoietic populations and the role of human Ikaros in lineage commitment and proliferation, potentially giving insights into deviations that may occur in leukemiogenesis. In Specific aims 1 and 2, RNA PCR will be used to determine expression of Ikaros isoforms in FACS sorted cells at different stages of differentiation and cell cycle. Partitioning of Ikaros proteins between cytoplasm and nucleus will be determined by confocal immunofluorescence microscopy and by immunoblottng proteins from subcellular fractionations. Changes in Ikaros isoform expression associated with ontogeny will be examined by comparing expression in cord blood and bone marrow. Candidate regulatory isoforms will be identified based on changes in expression or localization patterns that occur at key points in differentiation or at particular points in cell cycle. In Specific Aim 3 the effects of overexpressing Ikaros-1 (DNA-binding), Ikaros-6 (DNA-nonbinding) or other Ikaros isoforms (candidate regulators identified in Specific Aims 1 and 2) on lineage commitment, cell cycle status, and proliferation will be determined. Ikaros isoforms will be overexpressed using lentiviral vectors and by protein transduction via a protein transduction domain (PTD) that allows bacterially expressed proteins to enter eucaryotic cells and selectively localize to either the nucleus or cytoplasm. The effects of Ikaros isoform overexpression and localization on lineage commitment and proliferation will be analyzed using bulk and single cell cultures designed to assay lymphoid, myeloid, and erythroid potential. Flow cytometry will be used to track changes in division of cells and cell cycle status associated with overexpression and localization of Ikaros isoforms.